Single-Molecule Electronic Nucleic Acid Sequencing-by-Synthesis Using Tagged Nucleotides and Nanopore Constructs With past NIH funding, we developed a single molecule nanopore-based sequencing by synthesis (SBS) strategy (Nanopore SBS) that accurately distinguishes the four DNA bases by electronically detecting 4 different polymer tags attached to the 5?-phosphate-modified nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primed template and polymerase, the polymer tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. While the Nanopore-SBS approach already produces good quality sequences, further optimization and development are needed to increase sequencing accuracy, while maintaining the ability of our nanopore-based single-molecule electronic system to produce long reads in real time. In this proposal, we will design and synthesize novel tagged nucleotides and construct nanopore-polymerase conjugates to control the sequencing reaction speed and increase single-molecule sequencing accuracy substantially, achieving desired polymerase catalytic rates and more efficient and consistent tag capture by the pores. We will use high ratios of unincorporable-to-incorporable tagged nucleotides to perform Nanopore-SBS. This will provide ample time to register currents due to the 4 unique tags on the unincorporable A, C, G and T nucleotides which display template-dependent binding to the polymerase ternary complex but are not incorporated into the growing DNA strand, followed by a new current level due to a 5th tag on the incorporable nucleotide which marks the transition to the extension step. This effectively eliminates insertion and deletion sequence artifacts, increases accuracy, and will be especially advantageous in DNA homopolymer repeat regions. This approach allows detection of a single nucleotide binding event multiple times (stutters) before the actual incorporation event, overcoming the inherent limitation of single molecule detection methods that only allow one chance for measurement. After optimizing the system with synthetic DNA templates, circular DNA libraries will be generated from viral and bacterial genomes to test this sequencing approach. With the improved tagged nucleotides, better regulated reaction kinetics, and newly designed polymerase-pore complexes, we will test the accuracy of our system on the nanopore arrays by sequencing these libraries at high coverage and comparing the results with other sequencing systems.